Natural immunoglobulins have been known for many years, as have the various fragments thereof, such as the Fab, (Fab′)2, Fv and Fc fragments, which can be derived by enzymatic cleavage. A natural immunoglobulin consists of a Y-shaped molecule having two protein chains (heavy and light), and having an antigen-binding site towards the end of each upper arm, generally known as the variable region. The remainder of the structure, generally known as the constant region domain, mediates the effector functions associated with immunoglobulins.
Natural immunoglobulins have been used in assay, diagnosis and, to a more limited extent, therapy. However, such uses, especially in therapy, have been hindered by the polyclonal nature of natural immunoglobulins. The advent of monoclonal antibodies of defined specificity increased the opportunities for therapeutic use. However, most monoclonal antibodies are produced following immunization of a rodent host animal with the target protein, and subsequent fusion of a rodent spleen cell producing the antibody of interest with a rodent myeloma cell. They are, therefore, essentially rodent proteins and as such are naturally antigenic in humans, frequently giving rise to an undesirable immune response termed the HAMA (Human Anti-Mouse Antibody) response.
Many groups have devised techniques to decrease the immunogenicity of therapeutic antibodies. These techniques generally involve the use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the antibody molecule. Early methods involved production of chimeric antibodies in which an antigen-binding site comprising the complete variable domains of the rodent antibody is linked to constant domains derived from a human antibody. Methods for carrying out such chimerization procedures are now well known in the art. More recent chimerization procedures have resulted in heteroantibodies comprising both the variable region domain of the target specific antibody chimerized with the variable domains of an antibody specific for Fc receptor. See U.S. Pat. No. 6,071,517 (herein incorporated by reference).
Given that chimeric antibodies still contain a significant proportion of non-human amino acid sequences, i.e. the complete non-human variable domains, they may still elicit some HAMA response. Therefore, other groups developed humanized versions of antibodies wherein the complementarity determining regions (CDRs) of a rodent monoclonal antibody are grafted onto the framework regions of the variable domains of a human immunoglobulin. Winter (EP-A-0239400), for instance, proposed performing such an alteration by site-directed mutagenesis using long oligonucleotides in order to graft three complementarity determining regions (CDR 1, CDR2 and CDR3) from each of the heavy and light chain variable regions. Such CDR-grafted humanized antibodies are much less likely to give rise to a HAMA response than chimeric antibodies in view of the much lower proportion of non-human amino acid sequences that they contain.
Although humanized antibodies were less immunogenic than their natural or chimeric counterparts, many groups found that CDR grafted humanized antibodies demonstrated a significantly decreased binding affinity (e.g., Riechmann, et al. Nature 332:323-327 1988). For instance, Reichmann and colleagues found that transfer of the CDR regions alone was not sufficient to provide satisfactory antigen binding activity in the CDR-grafted product, and it was also necessary to convert a serine residue at position 27 of the human sequence to the corresponding rat phenylalanine residue. These results indicated that changes to residues of the human sequence outside the CDR regions, in particular in the loop adjacent to CDR1, may be necessary to obtain effective antigen binding activity. Even so, the binding affinity was still significantly less than that of the original monoclonal antibody.
More recently, Queen et al (U.S. Pat. No. 5,530,101, herein incorporated by reference) described the preparation of a humanized antibody that binds to the interleukin 2 receptor, by combining the CDRs of a murine MAb (anti-Tac) with human immunoglobulin framework and constant regions. The human framework regions were chosen to maximize homology with the anti-Tac MAb sequence. In addition computer modeling was used to identify framework amino acid residues which were likely to interact with the CDRs or antigen, and mouse amino acids were used at these positions in the humanized antibody. The humanized anti-Tac antibody obtained was reported to have an affinity for p55 of 3×109 M−1, which was still only about one-third of that of the murine MAb.
Other groups identified further positions within the framework of the variable regions (i.e. outside the CDRs and structural loops of the variable regions) at which the amino acid identities of the residues may contribute to obtaining CDR-grafted products with satisfactory binding affinity. See, e.g., U.S. Pat. Nos. 6,054,297 and 5,929,212, herein incorporated by reference. Still, it is impossible to know before-hand how effective a particular CDR grafting arrangement will be for any given antibody of interest.
Recently, it was shown that interleukin-9 (IL-9) plays a critical role in a number of antigen-induced responses in mice including bronchial hyperresponsiveness, epithelial mucin production, eosinophilia and elevated inflammatory cell counts in bronchial lavage, including T cells, B cells, mast cells, neutrophils and eosinophils and elevated serum total IgE, typifying the allergic inflammation associated with asthma. See Levitt et al., U.S. Pat. No. 6,261,559, herein incorporated by reference. Structural similarity has been observed for the human and murine IL-9 genes, suggesting that human IL-9 would be expected to play a similar role in the indication of asthmatic immune responses. IL-9 is expressed by activated T cells and mast cells, with the protein serving as a T cell growth factor and a cytokine that mediates the growth of erythroid progenitors, B cells, eosinophils mast cells, and promoting the production of mucin by lung epithelium.
Levitt and colleagues demonstrated that pretreatment of mice with polyclonal neutralizing antibodies to murine IL-9 resulted in the complete protection of mice from antigen challenge in a mouse asthma model. It would be useful for human patients suffering from diseases or conditions associated with IL-9 expression such as asthma if antibodies having a low immunogenicity and a high binding affinity for human IL-9 could be designed for use in human therapy. The present invention provides for such antibodies, and their use in treating conditions wherein modulation and/or inhibition of IL-9 activity is therapeutically beneficial, e.g., allergic conditions such as bronchial hyperresponsivness, and atopic allergy including asthma.